Two distinct compartments of the immune system have been identified: (i) the systemic, which comprises the bone marrow, spleen and lymph nodes, and (ii) the mucosal, which comprises lymphoid tissue associated with mucosal surfaces and external secretory glands (McGhee et al., 1992). Mucosal surfaces are associated with the gastrointestinal (GI), genitourinary (GU) and respiratory tracts. Each compartment is associated with both humoral (antibodies) and cell-mediated (cytotoxic T-cells) responses, however there are differences in the nature of the immune responses induced in each compartment. Antibodies associated with the systemic compartment are predominantly of the IgG isotype, which function to neutralize pathogens in the circulatory system. In contrast, antibodies in the mucosae are primarily secretory IgA (S-IgA), which function to prevent entry of the pathogen into the body via the mucosal surface (Lamm et al., 1992). Systemic immunity cannot prevent entry of pathogenic organisms at mucosal surfaces.
Successful systemic immunization (i.e., delivery of antigen to the systemic compartment) will induce systemic immunity but does not usually yield mucosal immune responses. In contrast, antigen delivered at mucosal surfaces triggers both mucosal (at local and sometimes at distant sites) and systemic responses (Haneberg et al., 1994, Gallichan and Rosenthal, 1995).
Most vaccines developed to date are delivered parenterally, for example by intramuscular (IM) or intradermal (ID) injection, and as such induce primarily systemic immunity. However, the combined mucosal surface area is more than 200 times greater than that of the skin and is the primary site of transmission of numerous infectious diseases. Therefore, current vaccination strategies permit the pathogen to enter the body and only fight it once it is in circulation. Infection and morbidity rates could be reduced if effective mucosal immunity could be induced. Furthermore, there is evidence that mucosal vaccines may have a broader age range of recipients. Finally, mucosal vaccines are often administered by non-invasive means (e.g., nose drops, nasal spray, inhaled nebulizer), thus they are easier and less expensive to administer, have less need for trained personnel and no risk of needle stick injury or cross contamination (for reviews see Mestecky et al., 1992, Staats et al 1994, O, Hagan 1994).
As mentioned above, the hallmark of mucosal immunity is local production of S-IgA antibodies. These constitute >80% of all antibodies in mucosae-associated tissues and are induced, transported and regulated by mechanisms quite distinct from those of the systemic response. IgA is of primary importance to the host defense because it acts not only to resist strict mucosal pathogens but also of the many microorganisms which initially colonize mucosal surfaces but subsequently cause systemic disease. There appear to be three sites of IgA mediated mucosal defense: (i) in the lumen, where S-IgA can neutralize viruses, bacterial toxins and enzymes, and act as a mucosal barrier to prevent viral attachment, microbial adherence and adsorption of antigen; (ii) within epithelial cells where dimeric IgA can bind to intracellular antigen; (iii) within the lamina propria where dimeric IgA can complex with antigen and the immune complex thus formed transported to the lumen (Lamm et al., 1992).
Many vaccines in development are composed of synthetic or recombinant antigens (peptides or polypeptides). These are considered safer than traditional attenuated or inactivated whole pathogens, however they are often poorly immunogenic and require adjuvants to enhance specific immunity. For systemic administration, aluminum precipitates (alum) may be added to the antigens to augment immune responses. Alum is currently the only adjuvant licensed for human use in most countries including the USA, however it is not suitable for delivery to mucosal surfaces. Therefore most mucosal vaccines used today contain live-attenuated organisms, and little success has been obtained with mucosal delivery of subunit vaccines.
Cholera toxin (CT) is the mucosal adjuvant most commonly used in animal models. CT is the primary enterotoxin produced by Vibrio cholerae. It is an 84 kilodalton polymeric protein consisting of two subunits, a monomeric A subunit and a pentameric ring shaped B subunit. The B subunit binds GM1 gangliosides at the surface of eukaryotic cells and enables insertion of the A subunit into the cytosol, where it ADP-ribosylates GTP-binding regulatory protein associated with adenylate cyclase (Spangler, 1992).
CT enhances antigen presentation by macrophages, epithelial cells and B cells, promotes differentiation and isotype switching in B cells, and has complex inhibitory and stimulatory effects on T-cell proliferation and lymphokine production (Snider, 1995). Some groups report that CT can selectively activate Th2-type CD4+ T cells while inhibiting Th1-type cells (Takahashi et al., 1996,) while others report activation of both TH1 and Th2-type CD4+ T cells (Hornquist and Lycke, 1993). Differences may be due to a number of factors including route of immunization and the nature of the antigen.
The Escherichia coli heat-labile enterotoxin (labile toxin, LT) is structurally and functionally closely related to CT, and has similar adjuvant properties (Lycke et al., 1992). LT can confer immunity to co-administered antigens that are on their own non-immunogenic when administered by mucosal routes; this adjuvant effect is noted whether LT is simply mixed with or is physically coupled to the antigen (Holmgren et al., 1993).
While very effective as mucosal adjuvants in animal models, CT and LT are highly toxic, and especially so in humans. Genetically detoxified mutants of both CT and LT have been developed by using site-directed mutagenesis, which, at least in animal models appear to be less toxic yet retain some adjuvanticity (e.g., LTK63 is LT with a single substitution at serine-63) (Rappuoli et al., 1995, Douce et al., 1994, Pizza et al., 1994, De Haan et al., 1996). Nevertheless, the level of adjuvanticity appears to be proportional to the level of retained toxicity, and thus there is a clear need for an alternative safe and effective mucosal adjuvant.